Human Papillomaviruses (HPVs) infect epithelial tissues of skin and mucosa. HPVs are small double-stranded DNA viruses which cause benign and malignant lesions in the epithelia. Infections are usually cleared by the immune system, however they can become persistent and eventually may develop into various cancers. To date, at least 205 different HPV types have been described, 12 of them (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) have been classified as Class 1A carcinogens, also termed as High-risk (HR) HPVs. Types 16 and 18 are most prevalent. Most common cancer associated with HPVs is the cervical cancer: ˜500 000 new cases per year mostly in developing countries, and 266 000 deaths were reported in 2008. Moreover, HPV16 and HPV18, have been found to be associated with about 70 invasive carcinomas of the uterine cervix, as well as cancers of the oropharynx, anus, and other mucosal tissues. Low-risk HPVs on the other hand are not usually connected with malignancy.
Current therapies to remove lesions caused by HPVs include various cytodestructive procedures and immunomodulatory molecules, for example imiquimod. Imiquimod is not specific for HPV, but is used to treat various skin diseases, including skin cancer, melanoma, molluscum contagiosum and others. In addition, there are three vaccines available against HPVs: Gardasil (types 6, 11, 16 and 18), Gardasil 9 (types 16, 18, 31, 33, 45, 52, 58, 6 and 11) and Cevarix (types 16 and 18). The main active components in of these vaccines are the L1 capsid proteins of HPV viruses. Moreover these vaccines are only prophylactic and in addition to this the availability of the vaccines is limited.
HPVs encode two main oncoproteins, E6 and E7, which modify cellular environment to be more suitable for HPV replication by inducing DNA synthesis and cell transformation. E6 and E7 have crucial roles in HPV-related cancer development.
The life cycle of HPVs is strictly dependent on cellular differentiation program. Infection starts by virus entry into undifferentiated basal epithelial cells (possibly stem cells) through micro-wounds. Infection is established by initiating initial amplification during which viral copy number reaches up to few hundred copies per cell. Stable maintenance phase is next, during this stage HPV genome replicates approximately once per cell cycle the viral copy number is constant. Viral gene expression is kept at very low levels. Vegetative amplification and assembly of the virions takes place in highly differentiated cells. To replicate its genome, HPVs largely depend on cellular proteins. They themselves only encode two replication proteins: E1 and E2. E1 is an ATP-dependent DNA helicase, which initiates replication from the non-coding region of HPV genome—URR (LCR). It also interacts with various cellular replication proteins to facilitate viral replication, for example Topoisomerase I, Replication Protein A (RPA) and Polymerase Alpha. For replication, E2 protein forms a complex with E1 and directs it to the replication origin. In addition, E2 is involved in regulation of viral gene expression, and it tethers HPV genomes to mitotic chromosomes for efficient segregation.
Cellular DNA is constantly attacked by exogenous or endogenous DNA-damaging agents such as UV-wavelengths or errors in replication. To cope with it, cells have very sophisticated ways to ensure integrity of DNA, to avoid potential mutations and tumorigenesis: DNA damage response network (DDR). DDR network is orchestrated by different kinases (ATM, ATR and DNA-PK) and damage in DNA is repaired by two major pathways: Homologous recombination (HR) and Non-homologous end-joining (NHEJ). In recent years it has become clear that many viruses are not only capable of activating DDR but they also benefit from its activation. HPV genomes are replicated in distinct foci in the cell's nucleus which contain cellular proteins necessary in DDR. By activating DDR, HPVs recruit replication proteins to these foci which in turn help to efficiently replicate viral genomes. HPV replication machinery uses Homologous recombination dependent replication during amplification of viral genome which results in accumulation of oligomerized molecules containing at least two genomes.
Important proteins in DDR network are Tyrosyl-DNA-phosphodiesterases Tdp1 and Tdp2 as well as PARP1(poly-(ADP-ribose)-polymerase 1). Topoisomerases as part of Top1/Top2 cleaving complex (Top1/2cc) are proteins that locally relax otherwise tightly packed nucleic acids which is required for replication and transcription. Several exogenous and endogenous DNA damaging agents, however cause entrapment of Top1/2cc-s. Entrapped cleaving complexes cause DNA breaks due to the collision of replication forks. Tdp1 is a key enzyme in repairing DNA damage caused by Top1cc-s. It hydrolyzes phosphodiester bond between Top1 and DNA, thereby releasing Top1cc. PARP1 (Poly(ADP-ribose) polymerase-1) is an enzyme that binds to DNA and catalyzes addition of ADP-ribose polymers (PAR) to its target proteins. These modifications regulate cellular localization and biological activities of various proteins involved in DDR. Recently it has been suggested that PARP1 plays a key role in Tdp1-dependent repair of Top1-induced DNA damage. Tdp1 inhibitors together with PARP1 and/or Topoisomerase 1 inhibitors have been considered as targets in cancer therapy.
There are different model systems available for studying various stages of HPV life cycle. Most of the work has been done in human primary epithelial keratinocytes. However, using these cells is relatively time consuming and expensive, especially for high-throughput screening to identify novel HPV inhibitors. In this disclosure a U2OS-based model system with dual-luciferase system was used to measure cell growth/toxicity of the compounds and HPV genome replication suitable for high-throughput screening (PCT/EP2016/057898). This system allows studying all three replication stages of various HPVs. The gene expression of HPVs in U2OS cells is almost identical to the one in keratinocytes, making it suitable for identification of new anti-HPV drugs. Moreover replication mechanism and replication intermediates of various HPV subtypes seem to be identical to the ones seen in different keratinocyte cell-lines.
Human Papillomaviruses are important pathogens responsible for great number of various cancer cases worldwide. Regardless of the two existing vaccines, there is a need for antivirals against HPV infection because vaccines are only preventive and other types of therapies have proven to be unsuccessful. So far there are no specific HPV inhibitors available. High-throughput screening (HTS) of available chemical libraries is widely used technique to identify new inhibitors against various pathogens. However, by now there has not been a suitable model system for HT-screening of HPV-inhibitors.
This disclosure provides solutions to the above described problems.